Carbon-11 radiolabeling of an oligopeptide containing tryptophan hydrochloride via a Pictet-Spengler reaction using carbon-11 formaldehyde

Article abstract of DOI:10.1002/psc.2546

A procedure for the synthesis of a¹¹C-labeled oligopeptide containing [1-¹¹C]1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid ([1-¹¹C]Tpi) from the corresponding Trp?HCl-containing peptides has been developed involving a Pictet-Spengler reaction with [¹¹C]formaldehyde. The synthesis of [1-¹¹C]Tpi from Trp and [¹¹C]formaldehyde was examined as a model reaction with the aim of developing a facile and effective method for the labeling of peptides with carbon-11. The Pictet-Spengler reaction of Trp and [¹¹C]formaldehyde in acidic media (TsOH or HCl) afforded the desired [1-¹¹C]Tpi in a moderate radiochemical yield. Herein, the application of a Pictet-Spengler reaction to an aqueous solution of Trp?HCl gave the desired product with a radiochemical yield of 45.2%. The RGD peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys] was then selected as a substrate for the labeling reaction with [¹¹C]formaldehyde. The radiolabeling of a Trp?HCl-containing RGD peptide using the Pictet-Spengler reaction was successful. Furthermore, the remote-controlled synthesis of a [1-¹¹C]Tpi-containing RGD peptide was attempted by using an automatic production system to generate [¹¹C]CH₃I. The radiochemical yield of the [1-¹¹C]Tpi-containing RGD at the end of synthesis (EOS) was 5.9±1.9% (n=4), for a total synthesis time of about 35min. The specific activity was 85.7±9.4GBq/μmol at the EOS.

Full text of DOI:10.1002/psc.2546

Research Article
Received: 8 May 2013
Revised: 15 July 2013
Accepted: 17 July 2013
Published online in Wiley Online Library: 15 August 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2546
Carbon-11 radiolabeling of an oligopeptide
containing tryptophan hydrochloride via a
Pictet-Spengler reaction using carbon-11
formaldehyde
Masayuki Hanyu,a Yuuki Takada,a,b Hiroki Hashimoto,a Kazunori Kawamura,a
Ming-Rong Zhanga and Toshimitsu Fukumuraa*
A procedure for the synthesis of a11C-labeled oligopeptide containing [1-11C]1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid
([1-11C]Tpi) from the corresponding Trp•HCl-containing peptides has been developed involving a Pictet-Spengler reaction
with [11C]formaldehyde. The synthesis of [1-11C]Tpi from Trp and [11C]formaldehyde was examined as a model reaction with
the aim of developing a facile and effective method for the labeling of peptides with carbon-11. The Pictet-Spengler reaction
of Trp and [11C]formaldehyde in acidic media (TsOH or HCl) afforded the desired [1-11C]Tpi in a moderate radiochemical yield.
Herein, the application of a Pictet-Spengler reaction to an aqueous solution of Trp•HCl gave the desired product with a
radiochemical yield of 45.2%. The RGD peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys] was then selected as a substrate for the labeling
reaction with [11C]formaldehyde. The radiolabeling of a Trp•HCl-containing RGD peptide using the Pictet-Spengler reaction
was successful. Furthermore, the remote-controlled synthesis of a [1-11C]Tpi-containing RGD peptide was attempted by using
an automatic production system to generate [11C]CH3I. The radiochemical yield of the [1-11C]Tpi-containing RGD at the end of
synthesis (EOS) was 5.9 1.9% (n = 4), for a total synthesis time of about 35 min. The specific activity was 85.7 9.4 GBq/μmol
at the EOS. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: Positron emission tomography (PET); 11C-labeled oligopeptides; [1-11C]-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid
([1-11C]Tpi); Pictet-Spengler reaction; [11C]Formaldehyde; Remote-controlled synthesis
Some low molecular weight oligopeptides have been consid-
ered as potential imaging agents because they possessed good
Introduction
Molecular imaging is an emerging technology that allows for the
visualization of the interactions between molecular probes and
biological targets. Positron emission tomography (PET), in
particular, is a useful modality that enables in vivo biological
information to be obtained in a noninvasive manner using a
variety of PET radiopharmaceuticals [1]. Short half-life positron
emitters, such as 11C (T1/2 = 20 min), 13N (T1/2 = 10 min), 15O
(T1/2 = 2 min), and 18F (T1/2 = 110 min), have generally been used
for PET molecular imaging studies [2]. It is possible to synthesize
PET probes that possess the same chemical structures as the
parent unlabeled molecules without altering their biological
activity because, with the exception of 18F, the positron-emitting
radionuclides described earlier can replace their stable analogues
in biomolecules. However, 15O and 13N have only ever been used
in simple compounds such as [15O]O2 and [13N]NH3, respectively,
because of the limitations imposed by their short half-lives. Carbon
is present in all organic molecules to such an extent that the intro-
duction of carbon-11 to a molecule does not modify its properties.
In addition, low β+energy of carbon-11, 0.96 MeV, promises a short
positron range in tissue, contributing to high spatial resolution PET
imaging [1,2]. Furthermore, the short half-life and rapid decay of
carbon-11 out of radioactivity allow for same-day imaging and
repeated investigations with either the same or different radio
tracers within a few hours. All of these properties represent
desirable options for longitudinal therapy monitoring.
permeability properties that could permit rapid access to the
target tissues. Radiolabeled peptides are becoming increasingly
important in nuclear oncology, where they are used in the
diagnosis and treatment of several different cancers [3–5]. One
of the main challenges of PET for radiochemists is the develop-
ment of rapid synthetic methods for the introduction of short-
lived positron-emitting radionuclides into the peptide of interest.
Several methods have been developed for the synthesis of
11C-labeled oligopeptides using [11C]CH3I. The 11C-labeled
peptides reported to date include four enkephaline-peptides
prepared by the reaction of [11C]CH3I with the corresponding
sodium-generated sulfide-anion in liquid ammonia [6], as well as
11C-labeled peptides derived from a chemoselective radiolabeling
strategy involving the formation of oximes via the reaction of
p-[11C]methoxy-benzaldehyde with aminooxy-functionalized
*
Correspondence to: Toshimitsu Fukumura, Molecular Probe Program,
Molecular Imaging Center, National Institute of Radiological Science, 4-9-1
Anagawa, Inage-ku, Chiba 263–8555, Japan. E-mail: t_fukumu@nirs.go.jp
a Molecular Probe Program, Molecular Imaging Center, National Institute of
Radiological Science, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
b Department of Radiology, School of Medicine, Yokohama City University, 3-9
Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
J. Pept. Sci. 2013; 19: 663–668
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

HANYU ET AL.
peptides [7]. Unfortunately, however, these complex synthetic
procedures invariably occur over long periods relative to the short
half-life of carbon-11, and this has severely limited their application.
The development of procedures amenable to the synthesis of
novel carbon-11 labeled agents for use as tracers in biomedical
research is important for moving the PET imaging technique
forward into the future.
measured using a NaI(TI) scintillation detector system (Ohyo Koken
Kogyo Co., ltd, Tokyo, Japan). Electrospray ionization mass spectros-
copy (ESI-MS) analyses were conducted on an Applied Biosystems
MDS SCIEX Q-TRAP 4000 (Tokyo, Japan).
Synthesis of the precursor for the radiosynthesis of cyclo
[Arg-Gly-Asp-D-Tyr-Lys([1-11C]Tpi)] ([11C]2)
[11C]Formaldehyde ([11C]CH2O) has been used as a carbon-11
labeling agent for compounds required in PET studies [8–10].
Several methods have been reported for the preparation of
[11C]CH2O from [11C]CH3OH by metal catalysis [11,12]. There have
also been several reports describing the enzyme-mediated syn-
thesis of [11C]CH2O [13]. Nader et al. [14] reported the use of
[11C]CH2O for the synthesis of [1-11C]1,2,3,4-tetrahydro-β-
carboline-3-carboxylic acid ([1-11C]Tpi) (Figure 1) via a Pictet-
Spengler reaction [15,16]. The methods currently available for
the synthesis of [11C]CH2O used in the construction of PET com-
pounds, however, require particularly low temperatures (À52°C)
under rigidity control using lithium aluminum hydride [14]. For
these reasons, the use of [11C]CH2O has not been developed to
any great extent because the current labeling approaches using
[11C]CH2O are generally inaccessible. Hooker et al. [17] recently
reported the development of a facile method for the preparation
of [11C]CH2O. Furthermore, the treatment of tryptamine with [11C]
CH2O under acidic conditions provided [11C]2,3,4,9-tetrahydro-
1H-β-carboline in a good radiochemical yield. We envisaged that
the treatment of Trp with [11C]CH2O under acidic conditions
would provide [1-11C]Tpi as well as several related analogues.
Herein, we report the synthesis of [1-11C]Tpi-containing
oligopeptides via a Pictet-Spengler reaction using [11C]CH2O.
Cyclo[Arg-Gly-Asp-D-Tyr-Lys(Boc-Trp)] (4)
A 1 mol/l NaOH solution (100 μl) was added to a solution of cyclo
[Arg-Gly-Asp-D-Tyr-Lys] (12.3 mg, 20 μmol) and Boc-Trp-OSu
(10.1 mg, 25 μmol) in a mixture of water (250 μl) and DMF
(300 μl) at room temperature, and the resulting mixture was
stirred at room temperature for 6 h. The resulting crude products
were purified on a semi-preparative reversed-phase HPLC sys-
tem. Purification was performed on a YMC-Triart C18 column
(10 mm i.d. × 250 mm, 5 μm) (YMC, Kyoto, Japan). A flow rate of
5 ml/min was used with a linear mobile phase gradient starting
from 95% solvent A (0.1% TFA in water) and 5% solvent B (0.1%
TFA in MeCN) to 0% solvent A and 100% solvent B over a 40-
min run time. The absorbance was monitored at a wavelength
of 220 nm. The retention time for 4 was found to be 18.2 min,
whereas the retention time for cyclo[Arg-Gly-Asp-D-Tyr-Lys] was
for 8.63 min. The fractions containing the product were collected
and concentrated in vacuo to give 12.3 mg (67.9 %) of the
pure product. ESI-MS m/z found to be 906.3; calculated for
[C43H59N11O11 + H]+ requires 906.4.
Cyclo[Arg-Gly-Asp-D-Tyr-Lys(Trp)] hydrochloride (3•HCl)
Compound 4 (9.5 mg, 10 μmol) was added to a solution of 1 mol/l
HCl in AcOH (6 ml) and the resulting solution was allowed to
stand for 3 h at room temperature. The solution was then concen-
trated in vacuo and the crude product purified by semi-prepara-
tive HPLC. Purification was performed on a YMC-Triart C18
column (10 mm i.d. × 250 mm, 5 μm). A flow rate of 5 ml/min
was used with a linear mobile phase gradient starting from 95%
solvent A (0.1% TFA in water) and 5% solvent B (0.1% TFA in
MeCN) to 0% solvent A and 100% solvent B over a 40-min run
time. The absorbance was monitored at a wavelength of
220 nm. The retention time for 3 was found to be 11.3 min. The
fractions containing 3 were collected and concentrated in vacuo
to give a residue that was added to a 1 mol/l HCl solution (3 ml).
Removal of the solution gave the product as the corresponding
HCl salt (7.1 mg, 88.0%). ESI-MS m/z was found to be 806.6; calcu-
lated for [C38H51N11O9 + H]+ requires 806.3.
Materials and Methods
All of the commercial reagents and solvents used in this work
were used without further purification. 1,2,3,4-Tetrahydro-β-
carboline-3-carboxylic acid (Tpi), anhydrous N,N-dimethylformamide
(DMF), 1 mol/l NaOH, high performance liquid chromatography
(HPLC) grade MeCN and HPLC grade water were purchased
from Wako Pure Chemical Industries (Osaka, Japan). Boc-Tpi-OH
was purchased from Watanabe Chemical Industries, Ltd
(Hiroshima, Japan). Boc-Trp-OSu and 1 mol/l HCl in AcOH were
purchased from Kokusan Chemical Co., Ltd (Tokyo, Japan).
Cyclo[Arg-Gly-Asp-D-Tyr-Lys] was purchased from Peptides
International (Louisville, KY, USA). Trp hydrochloride was
purchased from Tokyo Chemical Institute Co., Ltd (Tokyo, Japan).
Sodium phosphate Corrective Injection 0.5mol/l was purchased
from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). Fetal
bovine serum was purchased from NICHIREI BIOSCIENCES INC.
(Tokyo, Japan). A dose calibrator (IGC-3R Curiemeter; Aloka,
Tokyo, Japan) was used for all of the radioactivity measurements
unless otherwise stated. HPLC analyses were performed using a
JASCO HPLC system (Tokyo, Japan). The effluent radioactivity was
Reference compound of cyclo[Arg-Gly-Asp-D-Tyr-Lys(Tpi)] (2)
Cyclo[Arg-Gly-Asp-D-Tyr-Lys(Boc-Tpi)] (5)
To a solution of Boc-Tpi-OH (5.5 mg, 17.4 μmol) and N-hydroxy
succinimide (2.2 mg, 19.1 μmol) in DMF (1.0 ml) was added N-
(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
(3.6 mg, 19.1 μmol), and the resulting mixture was allowed to
stand for 1 h at room temperature. A solution of cyclo
[Arg-Gly-Asp-D-Tyr-Lys] (7.2 mg, 11.6 μmol) in 1 mol/l NaOH
(100 μl) was then added to the reaction, and the resulting mix-
ture was allowed to proceed for 6 h at ambient temperature.
The crude product was then purified on a semi-preparative
reversed-phase HPLC system. Purification was performed on a
YMC-Triart C18 column (10 mm i.d. × 250 mm, 5 μm). A flow rate
of 5 ml/min was used with a linear mobile phase gradient
Figure 1. Structure of [1-11C]1,2,3,4-tetrahydro-β-carboline-3-carboxylic
acid ([1-11C]Tpi).
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 663–668

CARBON-11 RADIOLABELING OF AN OLIGOPEPTIDE
starting from 95% solvent A (0.1% TFA in water) and 5% solvent
B (0.1% TFA in MeCN) to 0% solvent A and 100% solvent B over
a 40-min period. The absorbance was monitored at a wave-
length of 220 nm. The retention time for 5 was found to be
20.5 min, whereas the retention time for cyclo[Arg-Gly-Asp-D-
Tyr-Lys] was 8.63 min. The fractions containing the product
were concentrated in vacuo to give 6.9 mg (65.3%) of the de-
sired product as an oil. ESI-MS m/z found to be 918.5; calculated
for [C44H59N11O11 + H]+ requires 918.4.
Radiolabeling of [11C]2 by Manual Synthesis
The [11C]CH2O/DMF solution formed earlier was added to a reac-
tion vial containing 3•HCl in water (200 μl). The reaction vial was
left open to the environment and the reaction mixture was
heated at 100°C for 5 min. The reaction mixture was then
quenched by the addition of water (500 μl). Aliquots of this mix-
ture were analyzed by reverse-phase HPLC by using a YMC-Triart
C18 column (4.6 mm i.d. × 150 mm, 5 μm). A flow rate of 1 ml/min
was used with a linear mobile phase gradient starting from 95%
solvent A (0.1% TFA in water) and 5% solvent B (0.1% TFA in
MeCN) to 0% solvent A and 100% solvent B over a 40-min period.
The absorbance was monitored at a wavelength of 280 nm.
Cyclo[Arg-Gly-Asp-D-Tyr-Lys(Tpi)] hydrochloride (2•HCl)
Compound 5 (4.9 mg, 5.3 μmol) was added to a 1 mol/l HCl/AcOH
(6 ml) and allowed to stand for 3 h at room temperature. The
resulting solution was subsequently concentrated in vacuo to
give the crude product, which was purified by semi-preparative
HPLC. The purification was performed on a YMC-Triart C18 col-
umn (10 mm i.d. × 250 mm, 5 μm). A flow rate of 5 ml/min was
used with a linear mobile phase gradient starting from 95% sol-
vent A (0.1% TFA in water) and 5% solvent B (0.1% TFA in MeCN)
to 0% solvent A and 100% solvent B over a 40-min period. The
absorbance was monitored at a wavelength of 220 nm. The re-
tention time for 2 was found to be 13.2 min. The fractions
containing 2 were collected and concentrated in vacuo to give
a solid residue, which was added to 1 mol/l HCl solution (3 ml).
Subsequent concentration of the solution in vacuo gave the de-
sired product as the corresponding HCl salt (3.9 mg, 90.0%). ESI-
MS m/z was found to be 818.7; calculated for [C39H51N11O9 + H]+
requires 818.9.
Radiolabeling of [11C]2 by Remote-Controlled Synthesis
For the remote-controlled synthesis of [11C]2, a versatile synthe-
sis system was used for multiple PET radiopharmaceuticals [18],
which was developed at the National Institute of Radiological
Sciences. The use of this system led to the formation of a
sequence program in accordance with the results from the man-
ual synthesis. The [11C]CH3I was bubbled into a reaction vial
containing a solution of trimethylamine N-oxide (2.0 mg) in
DMF (150 μl) at a flow rate of 30 ml/min at a temperature in
the range of À10 to À15°C. When the radioactivity reached a
plateau, the reaction vial was heated to 70°C with an air heater
and held at that temperature for 2 min. The valve for the
exhaust line was then turned on. Following the addition of a
3•HCl solution (16 μmol/200 μl), the valve for the exhaust line
was left open and the solution was heated at 100°C for 5 min.
A 0.1% H3PO4 solution (1 ml) was then added to the reaction vial
and the resulting mixture was passed through a 0.20 μm filter
(Millex-LG, 13 mm) before being placed into a 2 ml sample loop
on an injector valve. Purification was performed on an X-Bridge
C18 column (10 mm i.d. × 250 mm, 5 μm). A flow rate of 5 ml/min
was used with a linear mobile phase gradient starting from 95%
solvent A (0.1% H3PO4 in water) and 5% solvent B (0.1% H3PO4
in MeCN) to 0% solvent A and 100% solvent B over a 40-min
period. The eluent was monitored for its ultraviolet absorbance
(280 nm) and radioactivity. The peak corresponding to [11C]2
was collected into a rotary evaporator flask containing a 25%
ascorbic acid injection (100 μl). The solvent was removed
in vacuo at 150°C, and the resulting residue was dissolved in a
sodium phosphate buffer (3 ml, pH 6.5).
Preparation of [11C]CH2O
The [11C]CH2O for the radiosynthesis was prepared from [11C]CH3I
as previously described [17]. [11C]CO2 was produced by using the
14N(p,α)11C nuclear reaction in a 0.01% oxygen-containing nitro-
gen gas with 18 MeV proton beams. Following the bombardment
process, [11C]CO2 was transferred to a vessel containing a
50 mmol/l solution of lithium aluminum hydride in THF (500 μl)
at a temperature in the range À10 to À15°C. The resulting solu-
tion was concentrated to dryness and a 57% solution of HI
(400 μl) was added to the vessel. The mixture was then heated
to 150°C to produce [11C]CH3I. The [11C]CH3I was purified by pass-
ing it through a column composed of Ascarite II and P2O5 and
collecting the eluent into a reaction vial containing a solution of
trimethylamine N-oxide (4.0 mg) in DMF (300 μl) at room temperature
with N2 gas (30 ml/min). When the radioactivity in the reaction vial
reached a plateau, the gas stream was suspended and the resulting
solution was heated to 70°C for 2 min to give [11C]CH2O.
Radiochemical Purity and Specific Activity Determinations
The radiochemical purity was assayed by analytical HPLC. Ali-
quots of the final products were analyzed by reverse-phase HPLC
onto an X-Bridge C18 column (4.6 mm i.d. × 150 mm, 5 μm). A
flow rate of 1 ml/min was used with a linear mobile phase gradi-
ent starting from 95% solvent A (0.1% TFA in water) and 5%
solvent B (0.1% TFA in MeCN) to 0% solvent A and 100% solvent
B over a 40-min period. The absorbance was monitored at a
wavelength of 220 nm. The retention time for [11C]2 was found
to be approximately 12 min. The identity was confirmed by
co-injection with the 2. The peak lag on the chromatogram
corresponds to line length between ultraviolet detector and NaI
(TI) scintillation detector. The radiochemical purity for [11C]2
was found to be >98%, and the average specific activity was
determined to be 85.7 9.4 GBq/μmol.
Radiolabeling of [1-11C]Tpi ([11C]1) by manual synthesis
The [11C]CH2O/DMF solution formed earlier was added to a reac-
tion vial containing a Trp (Trp•HCl) solution (200 μl). The reaction
vial was left open to the environment and the reaction mixture
was heated at 100°C for 5 min. The reaction mixture was then
quenched by the addition of water (500 μl). Aliquots of this mix-
ture were analyzed by reverse-phase HPLC by using a SunFire
C18 column (4.6 mm i.d. × 150 mm, 5 μm) and a mobile phase
consisting of MeCN and 50 mM HCOONH4 (1 : 4). A flow rate of
1.0 ml/min was used and the absorption was monitored at a
wavelength of 254 nm. The retention time for [11C]1 was found
to be approximately 5.6 min.
J. Pept. Sci. 2013; 19: 663–668 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

HANYU ET AL.
Stability of [11C]2
of an acid catalyst to give the desired product in a 45.2% radio-
chemical yield (Table 1, entry 5). In contrast, the reaction
between [11C]CH2O and Trp in a mixture of DMF and water
was unsuccessful (Table 1, entry 6). Thus, when Trp•HCl was
used in the Pictet-Spengler reaction, it effectively provided the
acid catalyst under trace amounts of [11C]CH2O. The effects of
the temperature and the reaction time on the outcome of the
reaction were also evaluated, and found to be important
(Table 1, entries 7 and 8). In the current study, the Pictet-
Spengler reaction between [11C]CH2O and Trp•HCl was found
to proceed in the absence of an additional acid catalyst when the
materials were heated in aqueous DMF. This procedure represents
an effective radiolabeling method because it requires particularly
mild conditions. The radiolabeling of bioactive oligopeptides
containing Trp•HCl with [11C]CH2O via a Pictet-Spengler reaction
could therefore be a useful procedure.
Cyclic RGD peptides, such as cyclo[Arg-Gly-Asp-D-Tyr-Lys], are
potent antagonists for the αvβ3 integrin receptor [21]. A variety
of different cyclic RGD peptides conjugated to a radioactive
tracer have been reported for the PET imaging of tumors that
overexpress the αvβ3 integrin receptor [22,23]. With this in mind
and to establish further potential uses for our new labeling
method, we investigated the application of our direct labeling
method using [11C]CH2O to the model cyclic RGD peptide cyclo
[Arg-Gly-Asp-D-Tyr-Lys(Trp)] hydrochloride (3•HCl). The substrate
of 3•HCl was prepared via the conjugation of Boc-Trp-OH with
the model cyclic RGD peptide according to the activated ester
method under slightly basic conditions, followed by the
deprotection of the Boc group with a solution of 1 mol/l HCl/
AcOH. Compound 3•HCl was labeled with [11C]CH2O according
to the same procedure used for the synthesis of [11C]1. The
reaction of compound 3•HCl with [11C]CH2O under the conven-
tional manual synthetic procedure proceeded smoothly to give
the desired product cyclo[Arg-Gly-Asp-D-Tyr-Lys(1-[11C]Tpi)]
([11C]2) with a radiochemical yield of 22.3 4.3% (not decay-
corrected), as shown in Figures 2 and 3. Interestingly, the
guanidino, phenolic hydroxy, carboxylic acid and amide groups
of cyclic RGD peptide remained intact under the reaction con-
ditions. Although this method needs an oligopeptide having
Trp residue on the terminal site except for the C-terminal side,
a Trp residue can be easily introduced to the epsilon amino
group of Lys residue or N-terminal amino acid residue. Based
on this result, it is therefore clear that this procedure could be
particularly effective for the direct formation of cyclic C-C
bonds for the radiolabeling of oligopeptides without the need
for protecting groups.
The approximately 20 MBq of [11C]2 was added to a reaction vial
containing a fetal bovine serum (100 μl). The reaction mixture
was incubated at 37°C. After 60 min, the mixture was then
quenched by the addition of MeCN (200 μl), and then centrifuged
at 12 000 revolutions per minute for 4 min. Stability was analyzed
with reverse-phase HPLC system using a X-Bridge C18 column
(4.6 mm i.d. × 150 mm, 5 μm). A flow rate of 1 ml/min was used
with a linear mobile phase gradient starting from 95% solvent
A (0.1% TFA in water) and 5% solvent B (0.1% TFA in MeCN) to
0% solvent A and 100% solvent B over a 40-min period.
Results and Discussion
The synthesis of [1-11C]Tpi ([11C]1) was initially examined under
the traditional acid catalyzed Pictet-Spengler conditions using
Trp and a solution of [11C]CH2O in DMF. The radiosynthesis of
[11C]1 was conducted by mixing a solution of [11C]CH2O in DMF
with a solution TsOH in DMF, and using Trp instead of tryptamine
according to the previously described method [17]. The desired
product was obtained with a moderate radiochemical yield
(Table 1, entry 1). The reaction was then conducted by using an
aqueous solution of TsOH, because the Trp was poorly soluble
in the TsOH/DMF solution at room temperature. The Trp
dissolved well in the aqueous solution of TsOH at room temper-
ature. These reaction conditions provided a similar result (Table 1,
entry 2) to the initial conditions, indicating that the reaction be-
tween [11C]CH2O and Trp in the DMF/water solution did not have
any discernible impact on the radiolabeling of [11C]1. When the
reaction was performed in the presence of the acid catalyst Yb
(OTf)3 [20], similar results were achieved (Table 1, entry 3). There
was no discernible improvement in the radiochemical conversion
of ([11C]1) when Yb(OTf)3 was used as an acid catalyst.
The choice of solvent used in this reaction was found to be par-
ticularly important because the synthesis of [11C]1 from [11C]CH3I
occurred over two steps but was conducted in one-pot. Saha
et al. [19] reported the preparation of a tetrahydro-β-carboline
derivative via a Pictet-Spengler reaction using a 10% aqueous
TFA solution as a reaction solvent. Based on this report, we
proceeded to evaluate the Pictet-Spengler reaction using 1 mol/l
HCl as the reaction solvent (Table 1, entry 4). Under these reaction
conditions, the radiochemical yield of [11C]1 was found to be similar
to those reported earlier (Table 1, entries 1–3). Interestingly, the
Pictet-Spengler reaction was found to proceed smoothly when
an aqueous solution of Trp•HCl was used without the addition
Table 1. Survey conditions for the preparation of [1-11C]Tpi a
.
b
Entry
Acid
Solvents
Reaction temperature (°C)
Reaction time
Substrate
Radiochemical yield (n = 4 )
1
2
3
4
5
6
7
8
TsOH (0.10 mmol)
DMF
H2O
100
5 min
5 min
5 min
5 min
5 min
5 min
5 min
1 min
Trp
42.3 3.2 %
45.3 2.1 %
34.7 8.5 %
44.8 2.9 %
45.2 3.6 %
—
TsOH (0.10 mmol)
100
Trp
Yb(OTf)3 (0.02 mmol)
H2O
100
Trp
—
—
—
—
—
1 mol/l HCl
H2O
100
Trp
100
Trp•HCl
Trp
H2O
100
room temperature
100
H2O
Trp•HCl
Trp•HCl
—
H2O
24.3 3.3 %
Reaction condition: [11C]CH2O/DMF (37-370 MBq) 200 μl; substrate (15 μmol); solvents 200 μl.
Determined by a radiochromatogram of the analytical high performance liquid chromatography.
a
b
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CARBON-11 RADIOLABELING OF AN OLIGOPEPTIDE
Figure 2. Radiolabeling method via the Pictet-Spengler reaction using [11C]CH2O.
[
11C]2
ascorbic acid
3
2
[
11C]2
2
0
2
4
6
8
10
12
14
16
Time (min)
0
2
4
6
8
10
12
14
16
Time (min)
Figure 5. Analytical high performance liquid chromatography chro-
matogram of co-injection of authentic sample with the purified [11C]2.
The black line represents the radioactive peak, and the black dotted line
shows the ultraviolet peak at 220 nm.
Figure 3. High performance liquid chromatography chromatogram: co-
injection of an authentic sample of 2 with the reaction mixture. The black
line represents the radioactive peak, and the black dotted line shows the
ultraviolet peak at 280 nm.
[
11C]2
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
16
Figure 6. Analytical radio HPLC chromatogram of purified [11C]2 (the
black dotted line) and [11C]2 after 60 min incubation with fetal bovine
serum (the black line).
Time (min)
Figure 4. Preparative high performance liquid chromatography chro-
matogram of [11C]2. The black line represents the radioactive peak, and
the black dotted line shows the ultaviolet peak at 280 nm.
[11C]2 was produced as the main radioactive peak in the reaction
mixture. The one-pot synthesis of [11C]2 from [11C]CH3I was
succeeded using an automatic production system. From a
starting point in the range of 21.0-22.2 GBq for the [11C]CO2,
[11C]2 was obtained at the end of synthesis in the range of
0.8-1.4 GBq. The average time required for the synthesis was
Based on the reaction conditions determined in the current
study, we proceeded to investigate the remote-controlled
radiosynthesis of [11C]2 by using an automatic production system
to generate the [11C]CH3I. Figure 4 shows a representative HPLC
chromatogram for the separation. This chromatogram shows that
J. Pept. Sci. 2013; 19: 663–668 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

HANYU ET AL.
6 Någren K, Ragnarsson U, Långström B. Synthesis of three 11C-labelled
methionine-containing enkephalin analogues. J. Labelled Compd.
Radiopharm. 1987; 15: 149–160.
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found to be 35 min from the end of the bombardment. The iden-
tity of [11C]2 was confirmed by its co-injection with compound 2
on the analytical HPLC system, as shown in Figure 5. The
radiochemical purity of [11C]2 was found to be greater than
98% and its specific activity was 85.7 9.4 GBq/μmol. Stability
of [11C]2 in fetal bovine serum was investigated by incubation
at 37°C for 60 min. The radiochemical purity of [11C]2 remained
>98%, as shown in Figure 6. The [11C]2 was radiochemically
stable toward in vitro degradation for the time of one PET scan.
10 Langer O, Krcal A, Schmid A, Abrahim A, Minetti P, Celona D, Roeda D,
Dollé F, Kletter K, Müller M. Synthesis of 1,1’[11C]-methylene-di-(2-
naphtol)([11C]ST1859) for PET studies for humans. J. Labelled Compd.
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Conclusion
In conclusion, we have successfully achieved the preparation of
11C]2 via a Pictet-Spengler reaction. This labeling reaction was
11 Roeda D, Crouzel C. [11C]Formaldehyde revisited: considerable con-
current [11C]formic acid formation in the low temperature conversion
of [11C]carbon dioxide into [11C]formaldehyde. J. Labelled Compd.
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[
completed under mild reaction conditions over a short reaction
time in only one step using the HCl salt of the precursor having
Trp on the terminal site, except for C-terminal side, without the
need for a protecting group. In addition, this labeling technique
could be used to increase the overall utility of 11C-labeled
oligopeptides as PET probes because this method allows for
the incorporation of carbon-11 into a cyclic C-C bond. This reac-
tion could be readily applied to an automated radiolabeling
platform using commercially available automated synthetic ap-
paratus for [11C]CH3I. The results obtained in the current study
can be extended to further studies aimed at the preparation
of 11C-labeled oligopeptides.
12 Roeda D, Dollé F. Preparation of [11C]formaldehyde using a silver-
containing ceramiccatalysis. J. Labelled Compd. Radiopharm. 2003;
46: 449–458.
13 Hughes JA, Jay M. Preparation of [11C]formaldehyde using a hollow
fiber membrane bioreactor. Nucl. Med. Biol. 1995; 22: 105–109.
14 Nader MW, Zeisler SK, Theobald A, Oberdorfer F. Low temperature
synthesis of No-carrier-added [11C]formaldehyde with metal hydrides
and preparation of [1-11C]1,2,3,4-tetrahydro-β-carboline derivatives.
Appl. Radiat. Isot. 1998; 49: 1599–1603.
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reaction in nature and in organic Chemistry. Angew. Chem. Int. Ed.
2011; 50: 8538–8564.
16 Van der May M, Windhorst AD, Klok RP, Herscheid JDM, Kennis LE,
Bischoff F, Bakker X, Heylen L, Jurzak M, Leysen JE. Synthesis and
biodistribution of [11C]R107474, a new radiolabeled α2-adrenocepter
antagonist. Bioorg. Med. Chem. 2006; 14: 4526–4534.
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method for the preparation of [11C]formaladehyde. Angew. Chem.
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Acknowledgements
We are grateful to Mr Y. Yoshida (SHI Accelerator Service Co., Ltd) for
his technical support with the remote-controlled radiolabeling. We
would also like to thank the staff of the Cyclotron Operation Section
and the Department of Molecular Probes of the National Institute of
Radiological Sciences (NIRS) for their support with the operation of
the cyclotron and the production of the radioisotopes. This work
was supported by JSPS-KAKENHI (Grant no. 24791357).
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wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 663–668